Preclinical Characterization of NGM936, a Novel Bispecific T Cell Engager Targeting ILT3 for the Treatment of Acute Myeloid Leukemia with Monocytic Differentiation

Abstract

Acute myeloid leukemia (AML) with monocytic differentiation [M4 and M5 AML per the French-American-British (FAB) classification] accounts for 25-30% of all AML cases and has a particularly poor prognosis, with a <15% five-year overall survival rate and an increased risk of bone marrow and extramedullary relapse after stem cell transplant compared with other AML subtypes. The use of T cell engagers in AML has been limited by the lack of suitable target antigens with high specificity for AML cells and minimal expression on healthy bone marrow progenitors. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is a highly specific marker of AML with monocytic differentiation and is not expressed on non-monocytic blood cells or hematopoietic progenitor cells. This highly restricted expression profile makes ILT3 a promising target for monocytic AML, with the potential to enable effective elimination of AML cells with less toxicity than current therapies. Here, we report the pre-clinical characterization of NGM936, an ILT3-based T cell engager for the treatment of monocytic AML and other ILT3+ hematologic malignancies. NGM936 is a monovalent scFv-Fab bispecific on an effectorless human IgG1 backbone (LALA) with a knob-in-hole Fc design for heterodimeric assembly. NGM936 demonstrates sub-nM affinity towards ILT3 of human origin (KD = 0.2 nM at 25°C by surface plasmon resonance) and is not cross-reactive towards other LILRA/B family members. A panel of more than 30 ILT3 x CD3 engagers in various formats was generated and screened in assays to measure both T cell-dependent cytotoxicity and cytokine release. NGM936 was identified for its ability to potently induce T cell-dependent cytotoxicity (TDCC) against ILT3+ AML cells while inducing minimal cytokine release. In both whole blood cytokine release assays and in TDCC assays in which cytokine secretion was measured after target engagement, induction of TNF-α, IL-6, IFN-γ, and IL-2 by NGM936 was considerably lower than that induced by a vibecotamab biosimilar (CD123 x CD3). NGM936 induced potent cytotoxicity when both expanded and naïve T cells were used as effectors. Importantly, NGM936 efficiently ablated tumor cells with a range of ILT3 expression, from ~1500 copies/cell to ~40,000 copies/cell. In addition, NGM936 failed to induce T cell-dependent cytotoxicity against CD34+ hematopoietic stem cells or non-monocytic immune cells, consistent with the lack of ILT3 expression on these cell types. In ex vivo cultures of primary M5 AML bone marrow, NGM936 induced a dose-dependent depletion of AML cells and a concordant increase in T cell proliferation and activation. Finally, NGM936 induced a dose-dependent depletion of circulating tumor cells in a xenograft model in which irradiated, immunodeficient NSG mice were engrafted with human PBMCs and human ILT3+ AML cells. NGM936 thus represents a promising new treatment strategy for monocytic AML, with the potential to eliminate monocytic leukemia cells while minimizing the myelotoxicity associated with ablation of healthy bone marrow.